Search

US-12618113-B2 - Genetic test kit for detecting thalassemia

US12618113B2US 12618113 B2US12618113 B2US 12618113B2US-12618113-B2

Abstract

One aspect of the invention is a method for amplifying alpha globin genes HBA1, HBA2 and HBA12 in a single PCR tube to determine an HBA genotype of a subject. This method employs five primers selected to accurate and sensitively identify the HBA1, HBA2, and HBA12, a gene found at a higher frequency in citizens of Saudi Arabia, by accurately annealing to nucleic acids in a biological sample and simultaneously amplifying sequences encoding the alpha globin genes. This invention includes a procedure and required reagents for the amplification of alpha globin genes in a single PCR tube.

Inventors

  • J. Francis BORGIO
  • Sayed ABDULAZEEZ
  • Fahd A. AL-MUHANNA
  • Amein Kadhem AL-ALI

Assignees

  • IMAM ABDULRAHMAN BIN FAISAL UNIVERSITY

Dates

Publication Date
20260505
Application Date
20240102

Claims (12)

  1. 1 . A genetic test kit, comprising: a primer pool composition comprising a set of primers for each of HBA1, HBA2 and HBA12, wherein the set of HBA1 primers comprises MA1F (SEQ ID NO: 3) and MA12R (SEQ ID NO: 4), wherein the set of HBA2 primers comprises MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO: 2 or 6) and wherein the set of HBA12 primers comprises MA12F (SEQ ID NO: 1) and MA2R (SEQ ID NO: 2 or 6); wherein at least one of said HBA1, HBA2 or HBA12 primers in the primer pool composition has been modified by conjugation to a fluorescent tag, biotin, quencher or other detectable reporter moiety and/or by substitution of a chemically modified, non-natural nucleotide for at least one natural nucleotide; and a polymerase chain reaction (PCR) tube, wherein the primer pool composition is present in the PCR tube.
  2. 2 . The genetic test kit of claim 1 , wherein the primer pool composition further comprises a human nucleic acid sample.
  3. 3 . The genetic test kit of claim 1 , wherein the primer pool composition further comprises a human nucleic acid sample, a DNA polymerase, dNTPs, a buffer solution, and bivalent cations and/or monovalent cations.
  4. 4 . The genetic test kit of claim 1 , wherein the primer pool composition comprises the set of HBA2 primers comprising MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO: 2).
  5. 5 . The genetic test kit of claim 1 , wherein the primer pool composition comprises the set of HBA2 primers comprising MA2F (SEQ ID NO: 5) and MA2R (SEQ ID NO: 6).
  6. 6 . The genetic test kit of claim 1 , wherein the primer pool composition comprises the set of HBA12 primers comprising MA12F (SEQ ID NO: 1) and MA2R (SEQ ID NO: 2).
  7. 7 . The genetic test kit of claim 1 , wherein the primer pool composition comprises the set of HBA12 primers comprising MA12F (SEQ ID NO: 1) and MA2R (SEQ ID NO: 6).
  8. 8 . The genetic test kit of claim 1 , wherein at least one of said HBA1, HBA2 or HBA12 primers in the primer pool composition has been modified by conjugation to a fluorescent tag.
  9. 9 . The genetic test kit of claim 1 , wherein at least one of said HBA1 , HBA2 or HBA12 primers in the primer pool composition has been modified by conjugation to biotin.
  10. 10 . The genetic test kit of claim 1 , wherein at least one of said HBA1, HBA2 or HBA12 primers in the primer pool composition has been modified by conjugation to a quencher.
  11. 11 . The genetic test kit of claim 1 , wherein at least one of said HBA1, HBA2 or HBA12 primers in the primer pool composition has been modified by substitution of a chemically modified, non-natural nucleotide for at least one natural nucleotide.
  12. 12 . The genetic test kit of claim 1 , wherein at least one of said HBA1, HBA2 and HBA12 primers in the primer pool composition has been modified by conjugation to a fluorescent tag, biotin, quencher or other detectable reporter moiety and/or by substitution of a chemically modified, non-natural nucleotide for at least one natural nucleotide.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS The present application is a Continuation of U.S. application Ser. No. 17/332,225, now U.S. Pat. No. 11,884,981, having a filing date of May 27, 2021 which is a Divisional of U.S. application Ser. No. 16/257,195, now U.S. Pat. No. 11,118,229, having a filing date of Jan. 25, 2019. REFERENCE TO A SEQUENCE LISTING In accordance with 37 CFR § 1.52(e)(5), the present specification makes reference to a Sequence Listing which is submitted herewith electronically as a .xml file named “551172US_ST26”. The .xml file was generated on Jan. 29, 2023 and is 22,447 bytes in size. The entire contents of the Sequence Listing are herein incorporated by reference. BACKGROUND Field of the Invention The invention pertains to the fields of medicine, genetics, and immunology. Description of Related Art The “background” description provided herein is for the purpose of generally presenting the context of the disclosure. Work of the presently named inventor(s), to the extent it is described in this background section, as well as aspects of the description which may not otherwise qualify as prior art at the time of filing, are neither expressly or impliedly admitted as prior art against the present invention. HBA Genes. The HBA1 and HBA2 gene sequences encode alpha globin proteins. The HBA1 gene provides instructions for making a protein called alpha-globin and this protein is also produced from a nearly identical gene called HBA2. These two alpha-globin genes are located close together in a region of chromosome 16 known as the alpha-globin locus. They are homologous in nature and show frequent intergenic exchange; Michelson, A. M.; and Orkin, S. H.; (1983). Boundaries of gene conversion within the duplicated human alpha-globin genes. Concerted evolution by segmental recombination. Journal of Biological Chemistry. 258(24): pp. 15245-15254; Law, H. Y.; Luo, H. Y.; Wang, W.; Ho, J. F.; Najmabadi, H. Ng. I S.; Steinberg, M. H.; Chui D. H.; Chong, S. S. (2006), Determining the cause of patchwork HBA1 and HBA2 genes: recurrent gene conversion or crossing over fixation events. Haematologica. 91(3):297-302; Hardison, R. C., (2012). Evolution of hemoglobin and its genes. Cold Spring Harbor perspectives in medicine. 2(12): p.a011627; Borgio et al., id. 2014; Borgio, id. 2015). HBA1 and HBA2 as well as their mutant forms have a high degree of molecular similarity and gene conversion between HBA1 and HBA2 in the alpha globin locus region is well documented and recently it has been discovered that the HBA2 has been replaced by a unique HBA12 gene conversion in 5.7% of the Saudi population. Direct sequencing of the HBA2 and HBA1 genes from 157 Saudi subjects revealed a new HBA2 gene conversion in cis or trans in 5.7% of these subjects. This new HBA2 gene convert is referred to as the α12 (HBA12) allele due to its combination of al (HBA1) and α2 (HBA2) sequences; see Borgio, J. F.; AbdulAzeez, S., Al-Nafie, AN., Naserullah, Z. A., Al-Jarrash, S., A novel HBA2 gene conversion in cis or trans: “alpha12 allele” in a Saudi population, Blood Cells, Molecules and Diseases. 53: 199-203 (2014) and Borgio, J. F., Molecular nature of alpha-globin genes in the Saudi population, Saudi Medical Journal 36:1271-1276 (2015). HBA12 Gene. The HBA12 gene allele comprises parts of the HBA1 gene (promoter, intron 1, coding region 2, intron 2) in its upstream region, while downstream of HBA12 gene is indistinguishable from the HBA2 gene (part of intron 2 and exon 3); Borgio et al., id. 2014, id.; Borgio, id. 2015. The HBA12 gene has the region starting −6 bp until 581 bp (3′ promoter, exon1, IVSI, exon2, and 5′IVSII) from HBA1 gene, and 774 bp (3′enhancer) onwards from HBA2 gene; and a total of 5.7% of the study population including sickle cell trait, hemophilia-A patient, SCD patients, and β-thal major patients were reported to have the new gene convert, α12 gene; Borgio, id. (2014). Reduced HBA2 in HBA12 patients. A reduced level of HBA2 was observed in the first five (HbScarrier; β-thalcarrier; β-thalmajor/α-thalcarrier; SCD+ve, and α-thalcarrier; HbScarrier α-thalcarrier) of six different subgroups of Saudis who carried HBA12; Borgio, et al., id. (2014). These groups as well as 32 different genotypes reported in the Saudi population are incorporated by reference to Borgio, et al., id. (2014). The presence of HBA12 genotypes α1α12/α1α12, α1−/α1α12, α1α2/α1α12, −α123.7/α1α12, and α1-4.2/α1α12 is associated with a reduced level of HBA2 (α2δ2) in β-thalassemia carriers, which might give a false negative result in conventional tests; Borgio et al., id., 2014. The HBA12 gene conversion presents challenges to conventional tests that only measure HBA1 or HBA2 and there is a need for a simple test that can be used for large scale screening of populations, like the Saudi population, where HBA12 is prevalent in order to proper asses the disease burden in these populations; Borgio, et al., id. (2015); Akhtar, M. S., Qaw, F., Borgio, J.